DESCRIPTION(adapted from the investigator's abstract): Ribosomal selection of aminoacyl-tRNA is a fundamentally important biological reaction. Recently, an "allosteric three-site model" was proposed as an alternative to the widely accepted proofreading mechanism for aa-tRNA selection at the ribosomal A site. In this model, aa-tRNA selection depends on a negative allosteric interaction between the ribosomal E (exit) and A sites. Deacylated tRNA in the E site antagonizes aa-tRNA selection at the A site such that only the cognate aa-tRNA binds strongly enough at the A site to disassociate the E site tRNA. There are data supporting and opposing this model. The goal of this project is to use an in vivo frameshift assay to determine whether the E site holds deacylated tRNA until an aa-tRNA is selected at the A site, as predicted by the allosteric three-site model. The assay is based on an experiment by van Duin in which tandem rare codons in a highly expressed mRNA were shown to promote frameshifting. This effect was proposed to be due to sequestration of the corresponding rare tRNA at the first rare codon in the ribosomal P site; the absence of aa-tRNA to bind at the second rare codon at the A site results in ribosomal stalling and a frameshift. If the allosteric model is correct, then overexpression of an mRNA containing the rare codons separated by an extra codon should also promote frameshifting, since the rare tRNA should remain sequestered in the E site in the absence of aa-tRNA to enter the A site. This prediction will be directly tested using appropriately modified frameshift test constructs; the results should demonstrate whether the E site does or does not effectively sequester tRNA in vivo, as predicted by the allosteric three-site model.